1) Field of the Invention
The present invention relates to a method for rapid isolation and purification of yolk antibodies, in particular IgY(xcex94Fc) antibody, from avian yolk, and the yolk antibodies produced thereby. More particularly, the present invention relates to a method for isolation and purification of yolk antibodies from avian yolk by an adsorption chromatographic procedure using a water insoluble non-charged absorbent to accomplish a desired separation of the aqueous and lipidic phases, and by a salting-out procedure that differentially precipitates the yolk antibodies. The present invention also relates to uses of the yolk antibodies in quantitative or qualitative immunoassay or in the preparation of pharmaceutical compositions directing to an etiological agent of interest.
2) Description of the Related Art
Antibodies are used widely in many biological investigations and clinical applications. Sera obtained from hyperimmunized mammalians are the most common source of polyclonal antibodies. Antibodies derived from such immune sera belong to a group of proteins called xe2x80x9cimmunoglobulins,xe2x80x9d among which the immunoglobulin G (IgG) is the most abundant. The IgG molecule consists of three domains, namely two Fab regions and one Fc region. The Fab portion involves mainly in antigen binding. The Fc portion, though having no ability to bind with an antigen, directs several biological activity of an antibody, such as complement fixing and Fc receptor binding.
In the art of immunodiagnostics, an intact IgG molecule is not suitable for use in detection systems and immunological assays involving mammalian sera since the Fc region on an IgG molecule is capable of binding to Fc receptors, activating the complement system, and reacting with rheumatoid factor in mammalian sera. Removal of the Fc portion of an IgG molecule frequently lead to a reduction in the interference (E. Lamoyi, Methods in Enzymology 121:652-663. (1986)).
Some of the suggested uses of antibody in immunotherapy include treating patients with intoxicated bacterial toxins or snake venoms (see, for example, U.S. Pat. Nos. 5,340,923 and 5,601,823), and protection of neonatal piglets against fatal enteric colibacillosis (see, for example, H. Brussow et al., J. Clin. Microbiol. 25:982 (1987); and C. O. Tacket et al., New Eng. J. Med. 318:1240 (1988)). Since the Fc fragment of an antibody molecule is known to be the most antigenic portion of the immunoglobulin (E. M. Akita et al., J. Immunol. Methods. 162:155-164 (1993)), cleavage of the same which results in the formation of an F(abxe2x80x2)2 fragment will reduce significantly a number of potential allergenic sites on the immunoglobulin molecule and is thus beneficial to human or an animal administered with the immunoglobulin.
Recently, the divalent F(abxe2x80x2)2 antibody fragment has been shown to be more useful in the immunodiagnostic tests (M. Muratsugu et al., J. Colloid Interface Sci 147:378 (1991); and J. L. Ortega-Vinuesa et al., J. Immunol Methods 90:29 (1996)) and more suitable for development of the immunoassays involving mammalian sera than the parent IgG.
The F(abxe2x80x2)2 antibody fragment, however, has not found widespread use in clinical immunodiagnostic kits as one might expect. This may be attributed to the difficulties and cost-ineffectiveness of large scale production of the F(abxe2x80x2)2 fragments, which is conventionally made by pepsin digestion of IgG and subsequent purification via chromatography.
Ducks and their phylogenetically close relatives and some reptiles, such as turtles, have three kinds of serum immunoglobulins: a macromolecular immunoglobulin IgM (800 kDa in duck), and two isoforms of low molecular weight IgG with sedimentation coefficients of 7.8S (in duck, 180 kDa) and 5.7S (in duck, 130 kDa), respectively. (E. R. Unanue et al., J. Exp. Med. 121:697-714 (1965); H. M. Grey, J. Immunol 98:811-819 (1967); and B. Zimmerman et al., Biochemistry 10:482-448 (1971)). Avian IgG is oftentimes called IgY due to their existence in egg yolk. The 5.7S IgY, constituted with shorter heavy chains, is structurally and antigenically similar to the F(abxe2x80x2)2 fragment of the 7.8S IgY (FIG. 1), and this fact leads to the nomenclature of IgY (equivalent to 7.8S IgY) and IgY(xcex94Fc) (equivalent to 5.7S IgY) to represent both isoforms of IgY (K. E. Magor et al., J. Immunol. 149:2627-2633 (1992)).
Studies conducted in the infected or experimentally immunized birds showed that duck antibodies are deficient in a number of biological effector functions, including complement fixation and Fc receptors binding, without sacrificing their binding activity to corresponding antigens (G. W. Litman et al., Immunochemistry 10:323 (1973); and T. E. Toth et al., Avian Dis. 25:17-28 (1981)). This may reasonably result from the apparent lack of Fc-equivalent region of the IgY(xcex94Fc) antibody that constitutes the quantitatively major component of duck antibody response. It is thus believed that the IgY(xcex94Fc) antibody, which appears to be a structural and functional analog of the F(abxe2x80x2)2 fragment, would provide magnificent advantages in immunological uses, if a promising process for manufacturing the antibody could be found, and the appropriate physical requirements for its activity could be identified.
Avian yolk antibodies have been reported to exhibit useful properties for both research and clinical applications as mammalian antibodies do (see, for example, U.S. Pat. Nos. 5,340,923; 5,585,098; 5,601,823; and 5,976,519). Egg yolks derived from a laying hen is inexpensive and more convenient and safer to handle as compared to the hyperimmunized mammalian sera. More importantly, yolk antibodies are able to stand up to the scrutiny under modern animal protection regulations (A. Poison et al., Immunol. Commun. 9:475 (1980); and B. Gottstein et al.). These facts suggest a potential use of egg yolk as a commercial source of antibodies.
However, high contents of lipidic substances, such as fatty acids, cholesterol and lecithin, in egg yolk make the isolation of yolk antibodies a cumbersome and laborious task. Many efforts have been made in this regard. For instance, water soluble precipitants, including agar, pectin (Japanese Kokai No. 64-38098 published in Feb. 8, 1989), dextran sulfate (J. C. Jensenius et al., J. Immunol. Methods 46:63 (1981)), natural gums (H. Hatta et al., J. Food Science 53:425(1988)) and polyethylene glycol (PEG) (A. Poison et al., Immunol. Invest. 14:323 (1985); see also U.S. Pat. No. 4,550,019 issued to A. Poison) were used to precipitate non-aqueous bio-molecules, mainly lipids and yolk granules, to thereby harvest a water soluble phase containing abundant yolk antibodies. A. Hassl et al. developed a two-step chromatographic process, comprised of hydrophobic interaction chromatography and size exclusive chromatography, for further isolation of yolk antibodies from a PEG-purified fraction (A. Hassl and H. Aspock, J. Immunol. Methods 110:225 (1988)). Akita et al. described an improved method for isolating IgY, in which yolk antibodies were extracted from chick eggs by diluting the egg yolks with a large volume of water and subjecting the resultant supernatant to size exclusive chromatography and/or ion exchange chromatography (E. M. Akita et al., J. Immunol. Methods. 160:207 (1993); and E. M. Akita and S. Nakai, J. Food Sci. 57:629 (1993)).
However, all these studies and patents focus on the isolation of the entire population of yolk antibodies (which includes at least IgY and IgY(xcex94Fc)) from avian eggs, rather than on the purification of IgY(xcex94Fc) antibody alone. Moreover, since IgY(xcex94Fc) antibodies are present only in birds belonging to the Order Anseriformes, including duck and goose, and since the lipid content in the egg yolk of the aniserform birds is reported higher than that in the galliform birds, such as chicken and turkey, the conventional methods described above provide no suggestion of a successful purification of IgY(xcex94Fc) antibody.
Therefore, there exists a need for a rapid, cost-effective and high-throughput process that provides easy isolation of the desired IgY(xcex94Fc) antibody from the antibody pool of avian egg while maintaining the activity of the IgY(xcex94Fc) antibody. The substantially purified IgY(xcex94Fc) antibody may acts as a new type of F(abxe2x80x2)2 antibody for various immunodiagnostic and immunotherapeutic uses.
An extensive research has been conducted to fulfill the industrial requirements for yolk antibodies as indicated above. It has now been unexpectedly found that a successful isolation of yolk antibodies from avian yolks can be readily accomplished through an adsorption chromatographic procedure using a water insoluble non-charged absorbent, and/or through a simple salting-out procedure that differentiates different isoforms of the yolk antibodies. According to the process of the present invention, the highly purified yolk antibodies, in particular the highly purified IgY(xcex94Fc), can be easily obtained with high yield in an economic manner, and are ready for a wide variety of immunological uses.
Accordingly, an object of the present invention is to provide a process for selectively isolating antibody isoforms from egg yolk, comprising the steps of:
(i) obtaining an aqueous fraction of egg yolk from a fowl hen egg;
(ii) a major portion of a first isoform of antibodies from the aqueous fraction of step (i) by salting out the aqueous fraction with a first non-denaturing salt of a first concentration; and
(iii) salting out a major portion of a second isoform of antibodies from the resultant aqueous fraction of step (ii) by adjusting the resultant aqueous fraction from step (ii) with a second non-denaturing salt of a second concentration;
with a proviso that when the first non-denaturing salt and the second non-denaturing salt are the same, the first and second concentrations are different.
Another object of this invention is to provide a process for isolating antibody from egg yolk, in which an absorbent is used to remove the majority of lipidic and caseinaceous matter normally present in egg yolk.
The present invention thus provides a process for isolating antibody from egg yolk, comprising the steps of:
(a) removing non-aqueous bio-molecules and granules from the egg yolk of a fowl hen egg to thereby obtain a water-miscible fraction containing yolk antibodies;
(b) passing the water-miscible fraction through a stationary phase containing an effective amount of a water insoluble non-charged absorbent capable of adsorbing water-miscible lipidic substances normally present in egg yolk;
(c) collecting the solution which has flowed through the stationary phase; and
(d) recovering the yolk antibodies from the flow-through solution of step (c).
The present invention also provides an alternative process for isolating antibody from egg yolk, comprising the steps of:
(a) removing non-aqueous bio-molecules and granules from the egg yolk of a fowl hen egg to thereby obtain a water-miscible fraction containing yolk antibodies;
(b) passing the water-miscible fraction through a stationary phase containing an effective amount of a water insoluble non-charged absorbent capable of adsorbing water-miscible lipidic substances normally present in egg yolk;
(c) eluting the stationary phase to obtain an eluate; and
(d) recovering the yolk antibodies from the eluate of step (c).
According to the process of this invention, an abundant amount of a selected isoform of yolk antibodies, in particular IgY(xcex94Fc) antibody, available for various industrial applications can be obtained in an economic, efficient and time-saving manner.
It is still another object of the invention to provide the clinical and research uses of the IgY(xcex94Fc) antibody so produced. In addition to the cost-effectiveness and ease of preparation, the IgY(xcex94Fc) antibody according to the present invention has advantages of being inactive to the complement system and rheumatoid factors in mammalian sera, and having poor cross-reactivity to mammalian IgG, and is thus particularly suitable for use in immunological assays involving mammalian sera with minimal interference. It would be appreciated by those skilled in the art that the IgY(xcex94Fc) antibody can be present in the form of a single reagent for clinical, research and other applications, or included in a commercial kit as an active component.
It is another specific object of the invention to provide a reagent for immunoassay of an etiological agent of interest, comprising an IgY(xcex94Fc) antibody obtained by the process according to this invention.